Heparin, a polydisperse, highly sulfated polysaccharide, is the most commonly used clinical anticoagulant. Despite the yearly use of over 50 million doses in the U.S. alone, heparin's exact chemical structure and the precise nature of its anticoagulant and antithrombotic activities remains unclear. Heparin anticoagulation results in frequent (8-33%) hemorrhagic complications, making it "the drug responsible for the most deaths in otherwise healthy patients" (Boston Collaborative Drug Surveillance Program). Recent clinical studies on low molecular weight (LMW) heparins (and heparinoids) demonstrate the dissociation of heparin's antithrombotic activities from its hemorrhagic side-effects. Despite this success, LMW heparins are still polydisperse, poorly characterized mixtures. The long term objective of this research is the preparation of pure structurally characterized LMW heparins with: high specificity; improved pharmacokinetics and bioavailability; and high antithrombotic activity with low hemorrhagic side-effects. The specific aims of this proposal are the following: 1. To screen a library of pure heparin fragments for their ability to bind to serine proteases serine protease inhibitors and for their ability to potentiate the inhibition of these proteases; 2. To assemble a manageable number of these fragments having both specific and non-specific binding affinities and (or) activities and to determine their chemical structure; 3. To test the activity of these pure heparin fragments using a variety of in vitro assays and to submit these to Dr. Fareed for evaluation as antithrombotic agents in vivo; 4. To examine both proteoglycan and raw heparin for the presence of these active sequences in order to relate these studies to heparin's "true biological functions." Heparin will be enzymatically depolymerized, fractionated by size and charge to obtain pure oligosaccharides and their activity (in vitro) and binding affinity to purified coagulation proteins will be measured. The structure of biologically interesting oligosaccharides will be determined by an integrated approach involving chemical, enzymatic and spectroscopic methods. SAR studies will use molecular graphics to compare the activities of a large number of structurally defined oligosaccharides. Direct comparison of the results obtained on clinically used commercial heparins with those obtained on proteoglycan heparins prepared from tissue will result in a better understanding of heparin's "true biological functions."